Investigating the phylogenetic history of toxin tolerance in mushroom‐feeding Drosophila

Abstract Understanding how and when key novel adaptations evolved is a central goal of evolutionary biology. Within the immigrans‐tripunctata radiation of Drosophila, many mushroom‐feeding species are tolerant of host toxins, such as cyclopeptides, that are lethal to nearly all other eukaryotes. In this study, we used phylogenetic and functional approaches to investigate the evolution of cyclopeptide tolerance in the immigrans‐tripunctata radiation of Drosophila. First, we inferred the evolutionary relationships among 48 species in this radiation using 978 single copy orthologs. Our results resolved previous incongruities within species groups across the phylogeny. Second, we expanded on previous studies of toxin tolerance by assaying 16 of these species for tolerance to α‐amanitin and found that six of them could develop on diet with toxin. Finally, we asked how α‐amanitin tolerance might have evolved across the immigrans‐tripunctata radiation, and inferred that toxin tolerance was ancestral in mushroom‐feeding Drosophila and subsequently lost multiple times. Our findings expand our understanding of toxin tolerance across the immigrans‐tripunctata radiation and emphasize the uniqueness of toxin tolerance in this adaptive radiation and the complexity of biochemical adaptations.

The genus Drosophila is comprised of ~2000 species, and it has been a model system for understanding the genetic basis of morphological changes and the selection pressures that underlie this variation (Markow, 2015(Markow, , 2019;;Markow & O'Grady, 2008).For instance, some well-studied morphological traits that vary among closely related species include wing patterning, body pigmentation, body size, and sexual dimorphism (Chown & Gaston, 2010;Koshikawa, 2020;Markow & O'Grady, 2005, 2008;Massey & Wittkopp, 2016;Williams & Carroll, 2009).Among Drosophila, biochemical adaptations are more challenging to study but just as variable as morphological traits.In particular, there is tremendous variation among species in what host the larvae develop.The model species Drosophila melanogaster is known for consuming rotting fruit in orchards, but across the Drosophila genus, species use tree sap, slime flux, fruit, cactus, mushrooms, and vegetation as developmental hosts (Powell, 1997).Some Drosophila species are generalists and can develop on many different hosts, whereas other species are specialized on a single host species.A suite of behavioral and biochemical traits is associated with host use, where each trait may have a separate genetic basis; for instance, adults must locate and then oviposit on the host, and the larvae must be able to develop successfully in the host (Anholt, 2020; Bernays & Chapman, 2007;Etges, 2019;Markow, 2019;Whiteman & Pierce, 2008).
Among the most fascinating of the host adaptations are Drosophila that develop on toxic hosts, as these species have evolved the ability to tolerate compounds that kill most other species.Most species of Drosophila that are tolerant to host toxins are specialist feeders, which is thought to reduce competition and predation (Jones, 2005).For instance, the cactophilic D. buzzatii is associated with the non-toxic prickly pear cactus (Opuntia sulphurea), whereas its sister species D. koepferae uses species within Trichocereus and Cereus genera that contain alkaloid secondary compounds (Hasson et al., 1992).Recent work has shown that enrichment of certain gene classes, such as responses to stress and metabolism of nitrogen compounds in columnar cacti dwellers like D. keopferae, may be responsible for the key adaptive differences between these species (Moreyra et al., 2023).Another example is D. sechellia, a specialist feeder on Morina citrifolia fruit (Louis & David, 1986), which contains octanoic acid, a fatty acid that is toxic to the closely related D. simulans (Furano et al., 2020;Jones, 2005;R'Kha et al., 1991).
Many Drosophila species utilize mushrooms as developmental hosts, and here we focus on the tolerance of certain Drosophila to cyclopeptides, which is the deadliest class of mushroom toxin (Bresinksy & Besl, 1990;Tang et al., 2016;Wieland, 1968).These toxins occur in some species of Amanita mushrooms, including the Death Cap (A. phalloides) and Destroying Angel (A. virosa), and are lethal to most eukaryotes, including humans.Fatalities are attributed to the amatoxin subclass, which act by binding to RNA polymerase II (RNAP II) and inhibiting mRNA production, leading to death (Lindell et al., 1970).Intriguingly, at least 12 Drosophila species in the immigrans-tripunctata radiation use mushrooms containing cyclopeptide toxins as developmental hosts (Scott Chialvo & Werner, 2018).A species is defined as 'tolerant' if they can survive from egg to adult eclosion on a laboratory diet containing 50 μg/g α-amanitin (Jaenike et al., 1983;Stump et al., 2011), though the toxic mushrooms these flies feed and develop on can contain up to 1600 μg of α-amanitin per gram of dried mushroom (Wieland, 1968).Studies have shown that while flies survive without visible impacts to fitness on the mean α-amanitin (250 μg/g) concentration, the extreme concentrations sometimes found in mushrooms (750-1000 μg/g) begin to affect the flies deleteriously (Jaenike, 1985).The tolerance of mushroom-feeding Drosophila to cyclopeptides is different from many other insect biochemical adaptations.First, typically only specialist feeders use hosts containing highly toxic compounds (Cornell & Hawkins, 2003;Whittaker & Feeny, 1971).Mushroom-feeding Drosophila, however, are generalists that use a variety of fleshy non-toxic and toxic fungi, and in some cases also use fruit and vegetation as developmental hosts (Jaenike & James, 1991;Lacy, 1984).Second, insects feeding on toxic hosts are often reported to be insensitive to the toxic compounds due to target site mutations (Holzinger & Wink, 1996;Karageorgi et al., 2019;Labeyrie & Dobler, 2004), but in toxin tolerant Drosophila, the target of amatoxins, RNAP II, does not contain mutations that prevent binding (Jaenike et al., 1983, Stump et al., 2011).It was found though that inhibition of Cytochrome P450s resulted in decreased tolerance in four of eight species assayed (Stump et al., 2011), suggesting P450s may be important to the mechanism of tolerance in some species.Third, the gut microbiome can contribute to detoxifying secondary metabolites encountered by some insects (Ceja-Navarro et al., 2015;Shukla et al., 2018).In one species of α-amanitin tolerant flies, however, alteration of the larval gut microbiome did not result in loss of tolerance (Griffin & Reed, 2020).Thus, these common mechanisms are not sufficient to explain toxin tolerance in mushroom-feeding

Drosophila.
Here, we study the evolution of cyclopeptide tolerance in mushroom-feeding Drosophila using phylogenetic and functional approaches.A first step to understand the emergence of a novel trait is to infer its phylogenetic history, meaning when it evolved in the lineages where it is present.This inference requires a well-resolved phylogeny.The evolutionary relationships in the immigrans-tripunctata radiation, where cyclopeptide tolerance occurs, are poorly understood and vary depending on the species sampled and data used to construct the phylogeny (Dyer et al., 2011;Finet et al., 2021;Hatadani et al., 2009;Morales-Hojas & Vieira, 2012;Perlman et al., 2003;Scott Chialvo et al., 2019;Spicer & Jaenike, 1996).Therefore, we first inferred the evolutionary relationships among an increased sampling of 48 species in the immigrans-tripunctata radiation.We then expand upon previous studies that examined the effect of α-amanitin (Jaenike, 1992;Jaenike et al., 1983;Stump et al., 2011) to characterize previously untested species' ability to survive on this toxin.We combine these empirical data with our species tree to construct a hypothesis of the ancestral state of toxin tolerance across this radiation.Our results suggest cyclopeptide tolerance arose once in the radiation and was then lost multiple times, and tolerance to α-amanitin is widespread throughout the radiation.

| Fly stocks, taxon sampling, and RNA sequencing
We maintained fly stocks used in phylogenetic analyses and toxin tolerance assays at 22.5°C with 50% relative humidity and a 12:12 light: dark cycle.Flies were maintained on 4-24 Instant Drosophila Media (Carolina Biological) with the addition of a piece of commercial mushroom (Agaricus bisporus), necessary for fly mating and development in these species, and a dental cotton roll.
The 48 species used in the phylogenetic analyses are listed in Table 1.The sampling includes all four testacea group species and 18 of the 26 quinaria group species.There are two strains of D. subquinaria, one inland (Hinton, Alberta) and one coastal (Portland, Oregon), as these populations are known to be strongly isolated (Jaenike et al., 2006).Members from the immigrans, tripunctata, and cardini groups were also included.The two outgroup species were D. grimshawi and D. virilis.The D. grimshawi, D. pruinosa, D. virilis, and D. quadrilineata genomes were obtained from the 101 Drosophilid Genomes Project (Kim et al., 2021).The D. innubila genome assembly was downloaded from NCBI (GenBank accession GCA_004354385.2).For the remainder of the species (Table 1), we generated transcriptomes.Flies were collected 24 h after emergence, and RNA was extracted using the Omega Bio-Tek EZNA Total RNA Kit 1 using an equal number of flash frozen males and females.
Samples were sent to NovoGene for library prep and sequencing.
Library preparation included poly-A enrichment, and libraries were sequenced as 150 bp paired-end reads.

| Transcriptome assembly and dataset generation
Transcriptomes were assembled using Oyster River Protocol v2.2.2 (MacManes, 2018) with default parameters.Each transcriptome and genome was assessed for completeness using the metazoan BUSCO v3.0.2 dataset v9 (Simao et al., 2015).We used TOAST (Wcisel et al., 2020) to generate an individual fasta file of each BUSCO ortholog, using the functions ParseBuscoResults and ExtractBuscoSeqs in R v3.6.1 (R Core Team, 2019).For species with genomes (Table 1), we extracted the corresponding sequence for each ortholog from the genomes using samtools faidx v1.10 (Li et al., 2009).These sequences were appended to the ortholog fasta files generated from TOAST.The new fasta files for BUSCO orthologs were run through TOAST's MafftOrientAlign, MissingDataTable, and SuperAlign functions to generate an alignment file for each of the 978 individual orthologs and a concatenated alignment file containing all orthologs (506,575,152 bp).

| Phylogenetic analyses
We generated species trees using maximum likelihood and coalescent-based frameworks.To generate a maximum likelihood tree, we used the concatenated dataset generated above.We ran Model Finder (Kalyaanamoorthy et al., 2017) in IQ-Tree v2.0.6 (Nguyen et al., 2015) to determine the model that best supported the data and generated a maximum likelihood species tree using that best-fit model.We assessed branch support with 100 bootstrap replicates.
Since the loci used and site rate variation can influence a tree topology, we performed a sensitivity analysis (Buddenhagen et al., 2016;Dowdy et al., 2020).Briefly, sites in each ortholog were placed into 10 bins based on evolutionary rate using TIGER v2.0 (Cummins & McInerney, 2011).The first bin contained invariant sites, the last bin the fastest-evolving sites, and the remaining sites were partitioned into the middle eight bins based on rate.
The program AMAS (Borowiec, 2016) was used to concatenate the binned sequences for each ortholog.For each ortholog, we created eight alignments by sequentially adding more rapidly evolving bins (e.g., 2 + 3, 2 + 3 + 4, etc.); the first bin was excluded since it only included invariant sites.Using RAxML v8.1.12(Stamatakis, 2014), maximum likelihood trees were generated for each alignment using the GTRCAT model and 100 bootstraps.We estimated the pairwise distance among trees using treeCMP v2.0.76 (Bogdanowicz et al., 2012).These trees were plotted using cmdscale in R v3.6.1 (R Core Team, 2019), and the Euclidean distance of each subsampled tree to their average center was calculated.We removed loci that did not have sites in all of the original site bins.The remaining 489 loci were then ranked based on this distance and placed equally into eight inclusion sets, from 61 to 489 loci, with each successive set containing more outlier trees.This was performed for each of the eight binning subsets to create a total of 64 locus-inclusion sets.New alignments were generated for each locus-inclusion set, and these were used to create a maximum likelihood tree for each locus in RAxML v8.1.12(Stamatakis, 2014) with bootstrap support values.These bootstrapped trees were used in ASTRAL-III v5.6.1 (Zhang et al., 2018) to identify support for the coalescent species tree for each locus-inclusion set.To assess support for the maximum likelihood tree, we concatenated the alignment for each inclusion set, and then used the RAxML-b flag to assess bootstrap support for each inclusion set.Heatmaps for each node of the tree were generated to show how support varies across these inclusion sets with the R package ggplot2.

TA B L E 1
Species used in the study and their respective species groups, along with strain ID and collection location.

| Toxin tolerance assays
Previous studies of Drosophila cyclopeptide tolerance (Jaenike et al., 1983;Spicer & Jaenike, 1996;Stump et al., 2011) characterized tolerance to ⍺-amanitin of 20 species distributed across six species groups in the immigrans-tripunctata radiation.To conduct a more comprehensive examination of the evolution of ⍺-amanitin, we assessed tolerance to ⍺-amanitin in 16 additional species from six species groups (Table 1).Tolerance assays were conducted in 7.5 mL glass scintillation vials containing 250 mg of a mixture consisting of 73.5% Instant Drosophila food and 26.5% ground freeze dried Agaricus bisporus mushroom resuspended in either 1 mL of water or 50 μg/g ⍺-amanitin (62.5 μg ⍺-amanitin in 1 mL water), which was the concentration used previously to identify tolerant species (Jaenike et al., 1983;Stump et al., 2011).A 1.5 cm × 4 cm piece of cotton watercolor paper was added as a pupation substrate.Each replicate consisted of placing early first instar larvae (15-25 per species; Table 1) into the vial and observing survival to adult for 30 days, and we conducted five replicate vials of each treatment.We coded the survival of each individual larvae to adulthood using a binary strategy (0 = Dead; 1 = Survived).We quantified the impact of ⍺-amanitin on survival for each species separately using a generalized linear model with a binomial distribution, logit link, and bias-adjustment using the brglm2 v0.8.2 package (Kosmidis, 2021).The only included model effect was toxin presence.Statistical analyses were completed in RStudio v2022.7.1.554.3 (RStudio Team, 2022).

| Ancestral state reconstruction of toxin tolerance
To analyze how toxin tolerance has evolved along the immigranstripunctata radiation, we inferred the ancestral state of toxin tolerance using RASP4 (Yu et al., 2020).This tool uses character state information for each species to infer the most likely ancestral state of each internal node of a phylogenetic tree.Data on toxin tolerance used for character state information is either from this study or previous studies (Jaenike et al., 1983, Spicer & Jaenike, 1996, Stump et al., 2011).These previous experiments were similar to ours.In Stump et al. (2011), feeding assays were performed in 4-dram vials containing either no toxin or 50 μg/g ⍺-amanitin, with first instar larvae added to the food.In Spicer and Jaenike (1996) and Jaenike (1985), tolerance was tested in 3-dram glass vials along a range of ⍺-amanitin concentrations, with eggs placed in the vials.In these studies, species that used toxic mushrooms as a developmental host in the wild survived on a concentration at or above 50 μg/g ⍺-amanitin.Our categories were (A) at least 10% absolute survival on diet with 50 μg/g ⍺-amanitin or (B) less than 10% absolute survival on diet with 50 μg/g ⍺-amanitin.We used the Bayesian Binary MCMC (BBM) analysis in RASP with a fixed Jukes-Cantor model and null root distribution for 5,000,000 generations using four chains sampled every 100 generations.The first 1000 samples were discarded as burn-in.We used only species for which we had physiological data from ⍺-amanitin and pruned the other species from the phylogeny, resulting in a phylogeny with 35 species.Ancestral state reconstruction was performed on both the coalescent and maximum likelihood phylogenetic trees.

| Phylogenetic inference
To resolve the species tree for the immigrans-tripunctata radiation, we generated transcriptomes or collected genomes for 48 species (Table 1).The final dataset contained 978 single-copy orthologs from the BUSCO metazoan database.If a locus was duplicated, we used the highest scoring sequence from the BUSCO output.For our

TA B L E 1 (Continued)
transcriptomes, the average number of single-copy orthologs was 67.1%, with a duplicate average of 30.4%.The genomes had an average of 96.3% single-copy orthologs and 1.8% average for duplicates.
The percentage of all BUSCO categories (Complete and single-copy, Complete and duplicated, Fragmented, and Missing) in each species are in Table 1.
We used maximum likelihood and coalescent methods to generate a species tree from the 978 orthologs.The best model for our maximum likelihood tree was GTR + F + R10.Both the maximum likelihood topology (Figure 1a) and coalescent analysis (Figure 1b) produced five main well-supported clades, which we refer to as clades Among deeper nodes, the relationships of Clades B, C, D, and E differ between the two phylogenies, with nearly all these basal nodes well supported in both trees.
We performed a sensitivity analysis to identify how the inclusion of faster-evolving loci affects support of our phylogenies and determines the best species tree for further analyses.The sensitivity analysis found substantial variability for support of internal nodes in our maximum likelihood tree (Figure 2a), with most nodes having much lower bootstrap support, especially those leading to clades C and D + E. Specifically, we find lower bootstrap support for intermediate bin combinations (4-6) at all sets of loci for many nodes.This can most likely be attributed to a large portion of the sites in the locus-inclusion set being largely conserved for bins 1-3, but the addition of more variable sites for intermediate bins (4-6) impacts tree reconstruction during bootstrapping.
When most sites are conserved in lower bins (1-3), bootstrapping is going to produce similar topologies each time; however, when some of the sites in the alignment become variable as they are in intermediate bins, the resampling done during bootstrapping could produce topologies that vary from the main tree because there is random variation at a portion of the sites.Bootstrap support is recovered when we include more sites (bins 7-9), likely due to some of the variable sites being shared between closely related species and the resampling during bootstrapping occurring with a larger number of sites that are a more equal mix between conserved and variable.
In contrast, the coalescent phylogeny produced consistently higher bootstrap support values for most nodes independent of how many loci were included (Figure 2b).A few internal nodes had variability in bootstrap support, but the inclusion of more loci and variable sites increased their bootstrap support.This suggests that to resolve a species tree, fewer loci are necessary using a coalescent approach, whereas maximum likelihood approaches require larger loci sampling with site rate variation to obtain the same level of resolution.Thus, we concluded the coalescent topology is the best supported species tree.

| Toxin tolerance
We assayed ⍺-amanitin tolerance in 16 species, representing six species groups, that had not previously been tested.We reared larvae on diets with and without ⍺-amanitin (50 μg/g) and measured the proportion that survived to adulthood (Figure 3, Table 1).
In six species, at least 10% of larva survived to adulthood on the diet with toxin; these include D. macrospina in the funebris group, D. guarani and D. subbadia in the guarani group, and D. occidentalis, D. suboccidentalis, and D. tenebrosa in the quinaria group.
Seven species produced no adults on ⍺-amanitin treatment, which

| Evolution of toxin tolerance
With the knowledge that toxin tolerance varies within the immigrans-tripunctata radiation, we sought to reconstruct the ancestral state of this trait.We examined toxin tolerance as a binary trait, using results from our toxin tolerance assays and previous studies (Jaenike et al., 1983;Spicer & Jaenike, 1996;Stump et al., 2011).We define tolerant as at least 10% survival at 50 μg/g of ⍺-amanitin and susceptible as less than 10% survival at this concentration.There is uncertainty on toxin ancestry for our deepest node in the tree, but nodes in the phylogeny leading to Clades B-E all indicate tolerance, which is subsequently lost at least five times (Figure 4).There

| DISCUSS ION
Species within the immigrans-tripunctata radiation of Drosophila exhibit a wide range of trait variation, including morphological characters, host preference, parasite prevalence, and toxin tolerance (Dombeck & Jaenike, 2004;Markow & O'Grady, 2008;Simunovic & Jaenike, 2006;Spicer & Jaenike, 1996;Stump et al., 2011;Werner et al., 2010).Of particular fascination is the tolerance found in many of these species to cyclopeptide toxins, as these are among the only eukaryotes known to consume and develop on these potent toxins (Scott Chialvo & Werner, 2018).Intriguingly, some species that are generalist feeders (i.e., on fleshy mushrooms, or a combination of mushrooms and rotting fruits and/or vegetative matter) are able to use cyclopeptide-containing mushrooms as developmental hosts (Jaenike et al., 1983;Lacy, 1984), while some other more specialized mushroom feeders in the radiation (i.e., D. funebris) are not (Korneyev, 2010;Stump et al., 2011).These patterns run contrary to general ecological patterns of toxin-tolerance being associated with host specificity.To broaden our understanding of cyclopeptide toxin tolerance and the evolutionary relationships within the immigrans-tripunctata radiation, we generated a transcriptome phylogeny, conducted survival assays, and reconstructed toxin tolerance in the radiation.
When attempting to understand how traits among a lineage evolve, it is imperative to ensure that sufficient loci and a large enough taxon sampling are used in tree-building.Recently, research on the superfamily Ephydroidea, which contains Drosophilidae, sought to tease apart inconsistencies with a broader sampling of species and increased number of nuclear genes (Winkler et al., 2022).
This work reaffirmed and supported the need for broad taxonomic sampling across a lineage to best understand the evolution of species and the traits that encompass them.Phylogenetic studies of the immigrans-tripunctata radiation have used either limited species sampling and/or few loci (Dyer et al., 2011;Hatadani et al., 2009;Izumitani et al., 2016;Scott Chialvo et al., 2019), resulting in incongruence among the recovered tree topologies.We reconstructed a species tree for 48 species in the immigrans-tripunctata radiation using maximum likelihood and coalescent phylogenetic approaches with 978 single-copy orthologs.Both trees produced the same five major clades, though there was variation in where they fell along the topology (Figure 1).Previous work using a smaller subset of this radiation could not confirm the monophyly of the quinaria group (Scott Chialvo et al., 2019).Our results support monophyly of the quinaria group, as the trees recovered from both analyses had the two subclades sister to one another (Figure 1).Interestingly, the testacea group (Clade D) has different locations along our topologies, either sister to Clade E (Figure 1a) or to the combined clades of B and C (Figure 1b).Due to these inconsistencies, we performed a sensitivity analysis and found the coalescent phylogeny produced the greatest support across all bin and loci combinations (Figure 2b).With factors such as incomplete lineage sorting giving high support to an incorrect topology generated with a concatenated loci dataset (Kubatko & Degnan, 2007;Mendes & Hahn, 2018;Roch & Steel, 2015), it is not surprising the coalescent topology was the most consistent.
We surveyed the ability of 16 species from six species groups to develop on diet containing α-amanitin, six of which have a greater than 10% survival rate (Figure 3).Three are members of the quinaria species group, which is known to include many tolerant species (Jaenike et al., 1983;Spicer & Jaenike, 1996;Stump et al., 2011).The   little is known about their natural history.In the funebris group, one of the three species tested, D. macrospina, survived at a high level on the ⍺-amanitin diet.This species breeds in woodlands near streams (Mainland, 1942;Miller et al., 2017), but nothing is known about its host usage.Another susceptible member of the group, D. funebris, is a specialized feeder on polypore, shelf fungi (Korneyev, 2010); this species did not produce any adults on the 50 μg/g α-amanitin diet.This is consistent with earlier studies of toxin tolerance in D. funebris (Stump et al., 2011).Interestingly, we find variable tolerance in the cardini group.For example, there is weak survival (<10%) on 50 μg/g α-amanitin diet for D. cardinoides, D. parthenogenetica, and D. nigrodunni.Drosophila acutilabella, another member of the cardini group, is known to feed on mushrooms and has higher survival on toxin food (21%) (Stump et al., 2011).Given that all previously tested α-amanitin-tolerant species use fleshy mushrooms as hosts (Jaenike et al., 1983;Lacy, 1984), our results suggest cyclopeptide tolerance is associated with this specific feeding behavior, and the presence of toxin tolerance in a species could serve as an indicator of its host usage.
Including our results, cyclopeptide tolerance has been identified in seven species groups in the immigrans-tripunctata radiation based on whether larvae can survive on a diet containing 50 μg/g ⍺-amanitin.While the 50 μg/g concentration is below the mean ⍺-amanitin concentration (250 μg/g) in toxic Amanita mushrooms (Tyler Jr. et al., 1966;Yocum & Simons, 1977), only species that consume fleshy mushrooms can survive on this concentration (Jaenike et al., 1983;Stump et al., 2011).Our findings indicate that cyclopeptide tolerance is more broadly distributed within the immigrans-tripunctata radiation than previously known, and our ancestral state reconstruction of toxin tolerance finds that α-amanitin tolerance evolved early in the radiation and was lost multiple times (Figure 4).
These results are nearly identical if we use the maximum-likelihood tree for trait reconstruction (results not shown).This is consistent with Spicer and Jaenike (1996), who proposed that toxin tolerance evolved once and was lost multiple times in the quinaria group.We note that Stump et al. (2011) showed that inhibition of Cytochrome P450s affected toxin tolerant species differently but did support tolerance as the ancestral state.
Unlike other Drosophila that specialize on a single toxic host, be associated with reduced parasitism (Jaenike, 1985).Specifically, the prevalence of nematode parasitism in flies collected from toxic mushrooms is significantly lower than in flies collected from nontoxic mushrooms, suggesting that nematodes are not tolerant of toxins.Thus, ⍺-amanitin-containing mushrooms provide a source of respite from nematode parasitism and the subsequent infertility that results, increasing their fitness (Jaenike, 1985).However, a study in D. putrida found no direct effects of ⍺-amanitin on the host-parasite interaction, suggesting that selection due to resource competition is a more likely mechanism to maintain tolerance to toxin (Debban & Dyer, 2013).Unlike many hosts, mushrooms are a patchy and ephemeral resource, and a newly emerged mushroom-feeding fly must find a new mushroom host promptly because its developmenhost has disintegrated.If hosts are limited, the ability to use both toxic and non-toxic mushrooms may be strongly favored.This tolerance is likely costly; however, as in all known instances where a species has switched from a mushroom to a non-mushroom host (i.e.
Our study characterized toxin tolerance using a single purified toxin, ⍺-amanitin, and one strain per Drosophila species; this did not capture any intraspecific variation in the trait.Other recent work examined four species in the immigrans-tripunctata radiation and found both intra-and interspecific variation in tolerance to a natural mix of toxins found in Amanita bisporus mushrooms (Kokate et al., 2022).The Kokate et al. (2022) study included D. neotestacea (testacea group), which has not been tested for tolerance to ⍺-amanitin specifically.With our expanded understanding of toxin tolerance across the immigrans-tripunctata radiation, future studies should examine tolerance of more species to a natural mix of toxins, as toxic Amanita mushrooms can contain over 10 different cyclopeptide toxins.Further, examining tolerance in multiple strains per species will further characterize intraspecific variation in this trait, refine ancestral state reconstruction of toxin tolerance, and the selective pressures that maintain this important adaptation.
In conclusion, the combination of our phylogenetic and functional approaches allowed us to disentangle previous incongruities in the phylogeny, as well as expand our understanding on toxin tolerance to ⍺-amanitin.Our results suggest that the evolution of toxin tolerance in mushroom-feeding Drosophila is ancestral in the immigrans-tripunctata radiation and was lost several times across the radiation.Future studies should expand from assaying tolerance with ⍺-amanitin to the full mix of toxins found in Amanita mushrooms to explore factors that contribute to the evolution and maintenance of tolerance within and among Drosophila species.

F I G U R E 4
Ancestral state reconstruction of toxin tolerance evolution for the coalescent analysis species tree.The ancestral state of toxin tolerance is indicated on each branch by the circles, with the probability indicated by the colors, as shown in the legend.We also denote losses of tolerance along the tree.

A
through E. Clade A consists of the immigrans group along with D. pruinosa and is sister to Clades B-E.Clade B is the funebris group.Clade C is the quinaria group and contains two sister clades within it (C1 and C2).Clade D is the testacea group and D. bizonata.Clade E represents the cardini and guarani groups, along with D. tripunctata, D. macroptera, and D. pallidipennis.Within each clade, species relationships are identical between the two topologies other than the relationship of D. brachynephros and D. phalerata within Clade C1.
is one loss of tolerance in the funebris group (Clade B) and two in the quinaria group (Clade C), including one in D. quinaria and another the common ancestor of D. deflecta, D. palustris, and D. subpalustris.Toxin tolerance is more variable in Clade E, where about half of the species assayed are susceptible.Tolerance was lost on the branch leading to D. pallidipennis and, for the cardini group, was either lost twice or lost once and then regained, and we suggest the former is most parsimonious.

F I G U R E 1
Species relationships for the immigrans-tripunctata radiation generated by (a) maximum likelihood or (b) coalescent methods.Branch labels indicate bootstrap support and are only shown for branches with less than 100% support.Clade A in yellow represents the immigrans group and D. pruinosa, Clade B in blue is the funebris group, Clade C in orange is the quinaria group, with two subclades marked C1 and C2, Clade D in purple is the testacea group and D. bizonata, and Clade E in green is the cardini and guarani groups along with D. tripunctata, D. macroptera, and D. pallidipennis.
Sensitivity analysis for the (a) maximum likelihood species tree and (b) coalescent analysis species tree.Legend inset shows a randomly chosen heatmap, which gives the number of loci (X-axis) and number of bins (Y-axis) used.The color gradient indicates bootstrap support for the branches based on the loci and bin combinations in the sensitivity analysis, with white being 0 bootstrap support (bs) and black 100 bootstrap support.
from the guarani and funebris species groups.The guarani group is neotropical(Penafiel-Vinueza & Rafael, 2018), and very most mushroom-feeding Drosophila are generalists on fleshy basidiomycetes and only a small portion of their diet is expected to consist of toxic mushrooms.The selective forces that maintain cyclopeptide tolerance in mushroom-feeding Drosophila remain unclear.One potential selective benefit is that toxin tolerance has been suggested to F I G U R E 3 Proportion of first instar larvae that survived to adulthood on diets containing 0 or 50 μg/g ⍺-amanitin.Species are clustered based on their species group and the error bars indicate the 95% binomial confidence intervals.
Note: ⍺-amanitin tolerance is defined as (a) survival at 50 μg/g ⍺-amanitin or (b) no survival at 50 μg/g ⍺-amanitin.Feeding behavior is described as M: mushrooms, V: decaying vegetation, and F: fermenting fruit.NT indicates 'not tested' as we do not have data for these.The type of assembly is listed as either transcriptome (T) or genome (G), with the number of reads included for species sequenced in this study.BUSCO v9 scores are based on the metazoan dataset.For species assayed for toxin tolerance in this study, we indicate the number of first-instar larvae placed in each vial, the larvae to adult survival in control and toxin treatments, and the results of the statistical analysis.Citations are as follows: a Stump et al. (2011), b Jaenike et al. (1983), c Spicer and Jaenike (1996), d Jaenike (1985), e Tuno et al. (2007), f Colón-Parrilla and Pérez-Chiesa (1999), g Werner and Jaenike (2017), h Kimura et al. (1977), i Werner and Jaenike (1995), j Shorrocks (1977), and k Kim et al. (2021).
These include D. arawakana, D. dunni, and D. similis in the cardini group, D. funebris and D. multispina in the funebris group, D. sulfurigaster in the immigrans group, and D. pallidipennis in the pallidipennis group.The remaining three species in the cardini group (D. cardinoides, D. ni-